Myogenesis is a favorable system to study the processes leading to the conversion of a multipotent precursor cell into one that has restricted its developmental potential to the myogenic lineage. The myoblasts resulting from this event, in turn, have the potential to express muscle-specific genes. However, in order to trigger this expression a second event needs to be triggered in response to environmentals cells, that leads to the expression of the differentiated phenotype. For these reasons, the long-term goal of this project continues to be to further our understanding of the molecular events leading to the production of the myogenic cell lineage, the commitment of myogenic cells to an irreversible post-mitotic state and the regulated expression of the muscle specific genes. Three main specific aims will be pursued: I). Using a combination of efficient gene transfer methods and sensitive selectable markers, we will isolate and characterize cDNA and genomic sequence, able to convert multipotent cells to the myogenic pathway. This approach to be used in this and the following section is based on two facts: a) unmethylated eukaryotic DNA (grown in bacteria) is active when transfected into competent cells and, b) there are several multipotent cell able to convert to the myogenic lineage upon the activation by demethylation with 5-agacytidine of what appears to be a single gene. The phenotype of these genes will be analyzed and its forced expression in a variety of cell studied to determine the basis for the mutual exclusivity observed between many differentiated phenotypes. II). Using similar approaches and the appropriate cell lines already at hand, we will isolate and characterize the gene(s) responsible for the commitment of myogenic cells to a terminal and irreversible differentiated phenotype. III). Using the DNA sequences and expression constructs already prepared we will further characterize the sequences and trans-acting factors responsible for the muscle-specific expression of the embryonic myosin heavy chain gene. Specific interest will be focussed on the role of a negative regulatory sequence that, when present, restricting the expression of this gene to differentiated muscle cells. It is expected that the work proposed will improve our understanding of the decision making events leading to the production of cell lineages and the eventual expression of a fully differentiated phenotype.